J Cell Biol, 2005 Jan 17, 168(2), 257 - 69 Role of mitochondria in the pheromone- and amiodarone-induced programmed death of yeast; Pozniakovsky AI et al.; Although programmed cell death (PCD) is extensively studied in multicellular organisms, in recent years it has been shown that a unicellular organism, yeast Saccharomyces cerevisiae, also possesses death program(s) . In particular, we have found that a high doses of yeast pheromone is a natural stimulus inducing PCD . Here, we show that the death cascades triggered by pheromone and by a drug amiodarone are very similar . We focused on the role of mitochondria during the pheromone/amiodarone-induced PCD . For the first time, a functional chain of the mitochondria-related events required for a particular case of yeast PCD has been revealed: an enhancement of mitochondrial respiration and of its energy coupling, a strong increase of mitochondrial membrane potential, both events triggered by the rise of cytoplasmic {Ca(2+)}, a burst in generation of reactive oxygen species in center o of the respiratory chain complex III, mitochondrial thread-grain transition, and cytochrome c release from mitochondria . A novel mitochondrial protein required for thread-grain transition is identified. J Cell Biol, 2005 Jan 17, 168(2), 209 - 19 Spindle-independent condensation-mediated segregation of yeast ribosomal DNA in late anaphase; Machin F et al.; Mitotic cell division involves the equal segregation of all chromosomes during anaphase . The presence of ribosomal DNA (rDNA) repeats on the right arm of chromosome XII makes it the longest in the budding yeast genome . Previously, we identified a stage during yeast anaphase when rDNA is stretched across the mother and daughter cells . Here, we show that resolution of sister rDNAs is achieved by unzipping of the locus from its centromere-proximal to centromere-distal regions . We then demonstrate that during this stretched stage sister rDNA arrays are neither compacted nor segregated despite being largely resolved from each other . Surprisingly, we find that rDNA segregation after this period no longer requires spindles but instead involves Cdc14-dependent rDNA axial compaction . These results demonstrate that chromosome resolution is not simply a consequence of compacting chromosome arms and that overall rDNA compaction is necessary to mediate the segregation of the long arm of chromosome XII. J Biol Chem . 2005 Jan 18; {Epub ahead of print} The role of the regulatory subunit of fission yeast calcineurin for in vivo activity and its relevance to FK506 sensitivity; Sio SO et al.; Calcineurin, a protein phosphatase required for Ca2+ signaling in many cell types, is a heterodimer composed of catalytic and regulatory subunits . The fission yeast genome encodes a single set of catalytic (Ppb1) and regulatory (Cnb1) subunits, providing an ideal model system to study these subunits' functions in vivo . Here, we cloned the cnb1+ gene, and showed that cnb1 knockout (Dcnb1) exhibits identical phenotypes with Dppb1, and that overexpression of Ppb1 failed to suppress the phenotypes of Dcnb1 . Interestingly, overexpression of C-terminal-deleted Ppb1 (Ppb1DC), the constitutively active form of Ppb1, also failed to suppress the phenotypes of Dcnb1 . FK506 caused MgCl2 sensitivity to the wild-type cells in an FKBP12-dependent manner . Co-overexpression of Ppb1 and Cnb1 suppressed the FK506-induced MgCl2 sensitivity, but the suppression was only partial, suggesting that an excess amount of Ppb1/Cnb1 complex cannot titrate out the FKBP12/FK506 complex . Although overexpression of Ppb1DC alone had little effect on cell growth, co-overexpression of Ppb1DC and Cnb1 caused distinct growth defect . FK506 suppressed the growth defect when Cnb1 was co-expressed using attenuated nmt1 promoter, but it failed to suppress the defect when Cnb1 was co-expressed using wild-type nmt1 promoter . Knockout of prz1+ gene, encoding a downstream target transcription factor of calcineurin, suppressed the growth defect irrespective of the promoter potency . These results suggest that Cnb1 is essential for the activation of calcineurin, and that the activated calcineurin is the pharmacological target of the FKBP12/FK506 complex in vivo. J Biol Chem . 2005 Jan 18; {Epub ahead of print} Search for apoptotic nucleases in yeast: Role of Tat-D nuclease in apoptotic DNA degradation; Qiu J et al.; DNA fragmentation/degradation is an important step for apoptosis . However, in unicellular organisms such as yeast, this process has rarely been investigated . In the current study, we revealed 8 apoptotic nuclease candidates in Saccharyomyces cerevisae, analogous to the Caenorhabditis elegans apoptotic nucleases . One of them is Tat-D . Sequence comparison indicates that Tat-D is conserved across kingdoms, implicating that it is evolutionarily and functionally indispensable . In order to better understand the biochemical and biological functions of Tat-D, we have overexpressed, purified and characterized the S . cerevisae Tat-D (scTat-D) . Our biochemical assays revealed that scTat-D is an endo-/exo-nuclease . It incises the double stranded DNA without obvious specificity via its endonuclease activity and excises the DNA from 3' to 5' end by its exonuclease activity . The enzyme activities are metal dependent with Mg++ as an optimal metal ion and an optimal pH around 5 . We have also identified three amino acid residues H185, D325, and E327 important for its catalysis . In addition, our study demonstrated that knockout of TAT-D in S . cerevisae increases the TUNEL positive cells and cell survival in response to hydrogen hyperoxide treatment, whereas overexpression of Tat-D facilitates cell death . These results suggest a role of Tat-D in yeast apoptosis. Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 497 - 501 Geotrichum silvicola sp . nov., a novel asexual arthroconidial yeast species related to the genus Galactomyces; Pimenta RS et al.; Four strains of an asexual arthroconidial yeast species were isolated from Drosophila flies in two Atlantic rain forest sites in Brazil and two strains from oak tasar silkworm larvae (Antheraea proylei) in India . Analysis of the sequences of the D1/D2 large subunit rRNA gene showed that this yeast represented a novel species of the genus Geotrichum, described as Geotrichum silvicola sp . nov . The novel species was related to the ascogenous genus Galactomyces . The closest relatives of Geotrichum silvicola were Galactomyces sp . strain NRRL Y-6418 and Galactomyces geotrichum . The type culture of Geotrichum silvicola is UFMG-354-2(T) (=CBS 9194(T)=NRRL Y-27641(T)). Gene, 2005 Jan 3, 344, 43 - 51 Epub 2004 Dec 10. Drag&Drop cloning in yeast; Jansen G et al.; We have developed a set of vectors that have enhanced capabilities for efficiently constructing and expressing differentially tagged fusion proteins using Drag&Drop cloning in the yeast Saccharomyces cerevisiae . The pGREG vectors are based on the pRS series with an additional general kanR selection marker . In vivo homologous recombination is used to introduce genes of interest into galactose-inducible expression vectors (pGREGs), permitting the formation of amino-terminal fusions . The vectors all contain common regions for recombination that flank the stuffer fragment . Introduction of common recombination sequences at the end of PCR fragments will permit the cloning of genes without the need for specific restriction sites . In this process, the selectable stuffer HIS3 gene is replaced by successful gene integration, and a screen for loss of the selection marker identifies potential recombinants . Due to the modular structure of the vectors, genes introduced into one vector can be readily transferred by in vivo recombination to all other members of the vector system, thus permitting rapid and easy Drag&Drop construction of a series of tagged proteins . The pGREG series combines features for expression, tagging, integration, localization and library construction with the advantage of obtaining immediate results from sub-sequent experiments . This Drag&Drop system also allows efficient cloning and expression of heterologous genes in large-scale experiments. FEBS J, 2005 Jan, 272(2), 524 - 37 Functional expression of olfactory receptors in yeast and development of a bioassay for odorant screening; Minic J et al.; The functional expression of olfactory receptors (ORs) is a primary requirement to examine the molecular mechanisms of odorant perception and coding . Functional expression of the rat I7 OR and its trafficking to the plasma membrane was achieved under optimized experimental conditions in the budding yeast Saccharomyces cerevisiae . The membrane expression of the receptor was shown by Western blotting and immunolocalization methods . Moreover, we took advantage of the functional similarities between signal transduction cascades of G protein-coupled receptor in mammalian cells and the pheromone response pathway in yeast to develop a novel biosensor for odorant screening using luciferase as a functional reporter . Yeasts were engineered to coexpress I7 OR and mammalian G(alpha) subunit, to compensate for the lack of endogenous Gpa1 subunit, so that stimulation of the receptor by its ligands activates a MAP kinase signaling pathway and induces luciferase synthesis . The sensitivity of the bioassay was significantly enhanced using mammalian G(olf) compared to the G(alpha15) subunit, resulting in dose-dependent responses of the system . The biosensor was probed with an array of odorants to demonstrate that the yeast-borne I7 OR retains its specificity and selectivity towards ligands . The results are confirmed by functional expression and bioluminescence response of human OR17-40 to its specific ligand, helional . Based on these findings, the bioassay using the luciferase reporter should be amenable to simple, rapid and inexpensive odorant screening of hundreds of ORs to provide insight into olfactory coding mechanisms. Genome Res . 2005 Jan 14; {Epub ahead of print} Genome-wide regulatory complexity in yeast promoters: Separation of functionally conserved and neutral sequence; Chin CS et al.; To gauge the complexity of gene regulation in yeast, it is essential to know how much promoter sequence is functional . Conservation across species can be a sensitive means of detecting functional sequences, provided that the significance of conservation can be accurately calibrated with the local neutral mutation rate . By analyzing yeast coding and promoter sequences, we find that neutral mutation rates in yeast are uniform genome-wide, in contrast to mammals, where neutral mutation rates vary along chromosomes . We develop an approach that uses this uniform rate to estimate the amount of promoter sequence under purifying selection . This amount is approximately 30%, corresponding to roughly 90 bp for a typical promoter . Furthermore, using a hidden Markov model, we are able to separate each promoter into distinct high and low conservation regions . Known regulatory motifs are strongly biased toward high conservation regions, while low conservation regions have mutation rates similar to that of the neutral background . Certain Gene Ontology groupings of genes (e.g., Carbohydrate Metabolism) have large amounts of high conservation sequence, suggesting complexity in their transcriptional regulation . Others (e.g., RNA Processing) have little high conservation sequence and are likely to be simply regulated . The separation of functionally conserved sequence from the neutral background allows us to estimate the complexity of cis-regulation on a genomic scale. Trends Cell Biol, 2005 Jan, 15(1), 10 - 8 Split decisions: coordinating cytokinesis in yeast; Wolfe BA et al.; Cytokinesis in eukaryotes involves the regulated assembly and contraction of a ring comprising filamentous (F)-actin and myosin II . Assembly of the contractile ring occurs through the accumulation of cortical cues at the specified division plane, followed by recruitment of F-actin, myosin II and accessory proteins involved in generating the mature ring . Ring contraction is temporally regulated to occur only after chromosome segregation and, in yeast, it is controlled by a conserved signaling cascade that becomes active only after Cdk1-Cyclin-B inactivation . In this article (which is part of the Cytokinesis series), we discuss recent studies that have begun to clarify both the spatial and the temporal order of ring assembly and that have illuminated the signals that trigger ring contraction in yeast . These studies add to the growing knowledge of the processes that control eukaryotic cell division. Cell, 2005 Jan 14, 120(1), 37 - 48 Mechanism of transcriptional silencing in yeast; Chen L et al.; Transcriptional silencing is a phenomenon in which the transcription of a gene by RNA polymerase II or III is repressed or not, dependent only on the gene's chromosomal location . Two prevailing models exist for silencing: (1) steric hindrance in silenced chromatin inhibits the binding of upstream activator proteins or polymerase or (2) silencing primarily blocks steps downstream of transcription preinitiation complex formation . Here, we test these models quantitatively for the case of SIR2-dependent silencing in budding yeast, using foreign and endogenous reporter proteins, at transgenic and endogenous loci . Our results contradict both models and show instead that transcriptional silencing at several URA3 transgenes, and at the naturally silenced endogenous HMRa and HMLalpha mating type genes, acts downstream of gene activator protein binding to strongly reduce the occupancy of TFIIB, RNA polymerase II, and TFIIE at the silenced promoters. Cytometry A . 2005 Jan 13; {Epub ahead of print} Detecting protein-protein interaction in live yeast by flow cytometry; Dye BT et al.; BACKGROUND: The yeast Saccharomyces cerevisiae is the most commonly used organism for studying protein- protein interactions . In this report we demonstrate the use of flow cytometry in observing fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent fusion proteins (CFP and YFP, respectively) as a marker for protein interaction in live yeast cells . Probability binning is also employed to provide a statistical confirmation of our observations . METHODS: We coexpressed CFP and YFP fusions containing the N-terminal transmembrane domain (NTM) of Tom70p in yeast and analyzed FRET in live cells with a multilaser flow cytometer . The Tom70p NTM was previously shown to be sufficient for mitochondrial localization and protein-protein interaction (Millar and Shore, 1994, J Biol Chem 269:12229-12232) . RESULTS: FRET was observed only in cells that expressed CFP and YFP fusions that each contained the wild-type NTM . The introduction of mutations previously shown to disrupt NTM interaction eliminated FRET . Probability binning confirmed that differences between the FRET channels of experimental and control samples were statistically and physiologically significant . CONCLUSION: Flow cytometric analysis of FRET in yeast is a powerful technique for studying protein-protein interactions . The use of flow cytometry allows FRET data to be gathered from a large number of individual cells, thus providing important advantages unavailable to other techniques . Its application to yeast presents a new method to a popular system widely used in proteomic studies . (c) 2005 Wiley-Liss, Inc. J Biol Chem . 2005 Jan 13; {Epub ahead of print} Activation of the iron regulon by the yeast Aft1/Aft2 transcription factors depends on mitochondrial, but not cytosolic iron-sulfur protein biogenesis; Rutherford JC et al.; Two transcriptional activators, Aft1 and Aft2, regulate iron homeostasis in Saccharomyces cerevisiae . These factors induce the expression of iron regulon genes in iron-deficient yeast, but are inactivated in iron-replete cells . Iron inhibition of Aft1/Aft2 is abrogated in cells defective for Fe-S cluster biogenesis within the mitochondrial matrix (Chen et al . J . Biol . Chem . 279, 29513) . To determine whether iron sensing by Aft1/Aft2 requires the function of the mitochondrial Fe-S export and cytosolic Fe-S protein assembly systems, we evaluated the expression of the iron regulon in cells depleted of glutathione and in cells depleted of Atm1, Nar1, Cfd1 and Nbp35 . The iron regulon is induced in cells depleted of Atm1 with Aft1 being largely responsible for the induced gene expression . Aft2 is activated at a later time in Atm1 depleted cells . Likewise, the iron regulon is induced in cells depleted of glutathione . In contrast, repression of NAR1, CFD1 or NBP35 fails to induce the iron regulon despite strong inhibition of cytosolic/nuclear Fe-S protein assembly . Thus, iron sensing by Aft1/Aft2 is not linked to the maturation of cytosolic/ nuclear Fe-S proteins, but the mitochondrial inner membrane transporter Atm1 is important to transport the inhibitory iron signal . Although Aft1 and Aft2 sense a signal emanating from the Fe-S cluster biogenesis pathway, there is no indication that the proteins are inhibited by direct binding of an Fe-S cluster. Biochem Biophys Res Commun, 2005 Feb 18, 327(3), 952 - 9 The yeast multidrug transporter Qdr3 (Ybr043c): localization and role as a determinant of resistance to quinidine, barban, cisplatin, and bleomycin; Tenreiro S et al.; Saccharomyces cerevisiae ORF YBR043c, predicted to code for a transporter of the major facilitator superfamily required for multiple drug resistance, encodes a plasma membrane protein that confers resistance to quinidine and barban, as observed before for its close homologues QDR1 and QDR2 . This ORF was, thus, named the QDR3 gene . The increased expression of QDR3, or QDR2, also leads to increased resistance to the anticancer agents cisplatin and bleomycin . However, no evidence for increased QDR3 expression in yeast cells exposed to all these inhibitory compounds was found . Transport assays support the concept that Qdr3 is involved, even if opportunistically, in the active export of quinidine out of yeast cell . A correlation was established between the efficiency of quinidine active export mediated by Qdr3p, Qdr2p or Qdr1p, and the efficacy of the expression of the encoding genes in alleviating the deleterious action of quinidine, as well as of the other compounds (QDR2>QDR3>>>QDR1). Curr Biol, 2005 Jan 11, 15(1), 68 - 72 Yeast Nhp6A/B and Mammalian Hmgb1 Facilitate the Maintenance of Genome Stability; Giavara S et al.; Saccharomyces cerevisiae Nhp6A and Nhp6B are chromatin architectural factors that belong to the high-mobility group box (HMGB) superfamily and appear to be functionally related to mammalian Hmgb1 . They bind to the minor groove of double-stranded DNA in a non-sequence-specific manner and thereby influence chromatin structure . Previous work has implicated these proteins in a variety of nuclear processes, including chromatin remodeling, DNA replication, transcription, and recombination . Here, we show that Nhp6A/B loss leads to increased genomic instability, hypersensitivity to DNA-damaging agents, and shortened yeast cell life span that is associated with elevated levels of extrachromosomal rDNA circles . Furthermore, we show that hypersensitivity toward UV light does not appear to reflect a decreased capacity for DNA repair but instead correlates with higher levels of UV-induced thymine dimer adducts being formed in cells lacking Nhp6A/B . Likewise, we show that mouse fibroblasts lacking Hmgb1 display higher rates of damage after UV irradiation than wild-type controls and also exhibit pronounced chromosomal instability . Taken together, these data indicate that Nhp6A/B and Hmgb1 protect DNA from damaging agents and thus guard against the generation of genomic aberrations. Biochem J . 2004 Dec 23; {Epub ahead of print} The yeast cyclin-dependent kinase inhibitor Sic1 and the mammalian p27(Kip1) are functional homologues with a structurally conserved inhibitory domain; Barberis M et al.; In Saccharomyces cerevisiae, Sic1, an inhibitor of cyclin-dependent kinase, blocks the activity of Cdk1/Clb5,6 (S-Cdk1) kinase required for DNA replication . Deletion of Sic1 causes premature DNA replication from fewer origins, extension of the S phase and inefficient separation of sister chromatids during anaphase . Despite the well documented relevance of Sic1 inhibition of S-Cdk1 for cell cycle control and genome instability, the molecular mechanism by which Sic1 inhibits S-Cdk1 activity remains obscure . In this report, we show that Sic1 is functionally and structurally related to mammalian cyclin-dependent kinase inhibitor (Cki) p27(Kip1) of the Kip/Cip family . A molecular model of the inhibitory domain of Sic1 bound to Cdk2/cyclin A complex suggested that the yeast inhibitor might productively interface with Cdk2/cyclin A mammalian complex . Consistently, Sic1 is able to bind to, and strongly inhibit the kinase activity of, Cdk2/cyclin A mammalian complex . Besides, the comparison of the different inhibitory patterns obtained using histone H1 or GST-pRb as substrates, the last one of which recognizes both the docking site and the catalytic site of Cdk2/cyclin A, offers interesting suggestions on the inhibitory mechanism of Sic1 . Finally, overexpression of the KIP1 gene in vivo in Saccharomyces cerevisiae rescues cell cycle-related phenotype of a sic1Delta strain, just as overexpression of the homologous SIC1 gene . Taken together, these findings strongly indicate that budding yeast Sic1 and mammalian p27(Kip1) are functional homologues with a structurally conserved inhibitory domain. Mol Biol Cell . 2005 Jan 12; {Epub ahead of print} Involvement of Sir2/4 in Silencing of DNA Breakage and Recombination on Mouse YACs during Yeast Meiosis; Klieger Y et al.; Monitoring Editor: Douglas Koshland Yeast artificial chromosomes (YACs) that contain human DNA backbone undergo DNA double strand breaks (DSBs) and recombination during yeast meiosis at rates similar to the yeast native chromosomes . Surprisingly, YACs containing DNA covering a recombination hot spot in the mouse Major Histocompatibility Complex class III region do not show meiotic DSBs and undergo meiotic recombination at reduced levels . Moreover, segregation of these YACs during meiosis is seriously compromised . In meiotic yeast cells carrying the mutations sir2 or sir4, but not sir3, these YACs show DSBs, suggesting that a unique chromatin structure of the YACs, involving Sir2 and Sir4, protects the YACs from the meiotic recombination machinery . We speculate that the paucity of DSBs and recombination events on these YACs during yeast meiosis may reflect the refractory nature of the corresponding region in the mouse genome. Mol Biol Cell . 2005 Jan 12; {Epub ahead of print} Golgi-to-Late Endosome Trafficking of the Yeast Pheromone Processing Enzyme Ste13p Is Regulated by a Phosphorylation Site in its Cytosolic Domain; Johnston HD et al.; Monitoring Editor: Benjamin Glick This study addressed whether phosphorylation regulates trafficking of yeast membrane proteins that cycle between the trans-Golgi network (TGN) and endosomal system . The TGN membrane proteins A-alkaline phosphatase, a model protein containing the Ste13p cytosolic domain fused to alkaline phosphatase (ALP), and Kex2p were found to be phosphorylated in vivo . Mutation of the S13 residue on the cytosolic domain of A-alkaline phosphatase to Ala was found to block trafficking to the prevacuolar compartment (PVC) while a S13D mutation generated to mimic phosphorylation accelerated trafficking into the PVC . The S13 residue was shown by mass spectrometry to be phosphorylated . The rate of ER-to-Golgi transport of newly synthesized A(S13A)-alkaline phosphatase was indistinguishable from wild-type indicating that the lack of transport of A(S13A)-alkaline phosphatase to the PVC was instead due to differences in Golgi/endosomal trafficking . The A(S13A)-alkaline phosphatase protein exhibited a TGN-like localization similar to that of wild-type A-alkaline phosphatase . Similarly, the S13A mutation in endogenous Ste13p did not reduce the extent of or longevity of its localization to the TGN as shown by alpha-factor processing assays . These results indicate that S13 phosphorylation is required for TGN-to-PVC trafficking of A-alkaline phosphatase and imply that phosphorylation of S13 may regulate recognition of A-alkaline phosphatase by vesicular trafficking machinery. Mol Biol Cell . 2005 Jan 12; {Epub ahead of print} The Roles of Fission Yeast Ase1 in Mitotic Cell Division, Meiotic Nuclear Oscillation and Cytokinesis Checkpoint Signaling; Yamashita A et al.; Monitoring Editor: Tim Stearns The Ase1/Prc1 proteins constitute a conserved microtubule associated protein family that is implicated in central spindle formation and cytokinesis . Here we characterize a role for fission yeast Ase1 . Ase1 localizes to microtubule overlapping zones, and displays dynamic alterations of localization during the cell cycle . In particular, its spindle localization during metaphase is reduced substantially, followed by robust appearance at the spindle midzone in anaphase . ase1 deletions are viable but defective in nuclear and septum positioning and completion of cytokinesis, which leads to diploidization and chromosome loss . Time-lapse imaging shows that elongating spindles collapse abruptly in the middle of anaphase B . Either absence or overproduction of Ase1 results in profound defects on microtubule bundling in an opposed manner, indicating that Ase1 is a dose-dependent microtubule bundling factor . In contrast microtubule nucleating activities are not noticeably compromised in ase1 mutants . During meiosis astral microtubules are not bundled and oscillatory nuclear movement is impaired significantly . The Aurora kinase does not correctly localize to central spindles in the absence of Ase1 . Finally Ase1 acts as a regulatory component in the cytokinesis checkpoint that operates to inhibit nuclear division when the cytokinesis apparatus is perturbed . Ase1, therefore, couples anaphase completion with cytokinesis upon cell division. Proc Natl Acad Sci U S A, 2005 Jan 18, 102(3), 707 - 12 Epub 2005 Jan 12. Rapid evolution of expression and regulatory divergences after yeast gene duplication; Gu X et al.; Although gene duplication is widely believed to be the major source of genetic novelty, how the expression or regulatory network of duplicate genes evolves remains poorly understood . In this article, we propose an additive expression distance between duplicate genes, so that the evolutionary rate of expression divergence after gene duplication can be estimated through phylogenomic analysis . We have analyzed yeast genome sequences, microarrays, and transcriptional regulatory networks, showing a >10-fold increase in the initial rate for both expression and regulatory network evolution after gene duplication but only an approximately 20% rate increase in the early stage for protein sequences . Based on the estimated age distribution of yeast duplicate genes, we roughly estimate that the initial rate of expression divergence shortly after gene duplication is 2.9 x 10(-9) per year, whereas the baseline rate for very ancient gene duplication is 0.14 x 10(-9) per year . Relative expression rate tests suggest that the expression of duplicate genes tends to evolve asymmetrically, that is, the expression of one copy evolves rapidly, whereas the other one largely maintains the ancestral expression profile . Our study highlights the crucial role of early rapid evolution after gene/genome duplication for continuously increasing the complexity of the yeast regulatory network. Yeast . 2005 Jan 11;22(2):99-110 {Epub ahead of print} A new mutation in the yeast aspartate kinase induces threonine accumulation in a temperature-regulated way; Velasco I et al.; In Saccharomyces cerevisiae, aspartate kinase (the HOM3 product) regulates the metabolic flux through the threonine biosynthetic pathway through feedback inhibition by the end product . In order to obtain a strain able to produce threonine in a controlled way, we have isolated a mutant allele (HOM3-ts31d) that gives rise to a deregulated aspartate kinase . This allele has been isolated as an extragenic suppressor of ilv1, which confers an Ilv(+) phenotype at 37 degrees C but not at 22 degrees C . We have stated that at high temperature the mutant aspartate kinase is slightly more deregulated and shows a higher specific activity, inducing threonine accumulation . The HOM3-ts31d allele carries a mutation that leads to a Ser399 --> Phe substitution in the postulated regulatory region of the enzyme . We have detected other changes in the nucleotide sequence but they are also present in the parental strain, reflecting the genetic differences between different wild-type strains . A sequence comparison among all the reported mutant aspartate kinases suggests that not all residues involved in regulation of the activity are clustered in the so-called regulatory domain, as is the case of that mutated in AK-R7, another deregulated aspartate kinase obtained with the same strategy of ilv1 suppression . Copyright (c) 2005 John Wiley & Sons, Ltd. J Biol Chem . 2005 Jan 11; {Epub ahead of print} Phosphate carrier has an ability to be sorted to either the TIM22 pathway or TIM23 pathway for its import into yeast mitochondria; Yamano K et al.; Most mitochondrial proteins are synthesized in the cytosol, imported into mitochondria via the TOM40 complex, and follow several distinct sorting pathways to reach their destination submitochondrial compartments . Phosphate carrier (PiC) is an inner membrane protein with 6 transmembrane segments (TM1-TM6) and requires, after translocation across the outer membrane, the Tim9-Tim10 complex and the TIM22 complex to be inserted into the inner membrane . Here we analyzed in vitro import of fusion proteins between various PiC segments and mouse dihydrofolate reductase . The fusion protein without TM1-TM2 was translocated across the outer membrane, but was not inserted into the inner membrane . The fusion proteins without TM1-TM4 were not inserted into the inner membrane, but instead translocated across the inner membrane . Functional defects of Tim50 of the TIM23 complex either by depletion of the protein or addition of anti-Tim50 antibodies blocked translocation of the fusion proteins without TM1-TM4 across the inner membrane, suggesting that lack of TM1-TM4 led to switch of its sorting pathway from the TIM22 pathway to the TIM23 pathway . PiC thus appears to have a latent signal for sorting to the TIM23 pathway, which is exposed by reduced interactions with the Tim9-Tim10 complex and maintenance of the import competence. FEBS Lett, 2005 Jan 17, 579(2), 559 - 66 Yeast two-hybrid screens imply that GGNBP1, GGNBP2 and OAZ3 are potential interaction partners of testicular germ cell-specific protein GGN1; Zhang J et al.; Gametogenetin (Ggn) is a testicular germ cell-specific gene specifically expressed from late pachytene spermatocytes through round spermatids . The function of gametogenetin protein 1 (GGN1) remains unknown . Here, we used the yeast two-hybrid approach to look for more GGN1 interacting proteins . We found that gametogenetin binding protein 1 (GGNBP1), gametogenetin binding protein 2 (GGNBP2) and ornithine decarboxylase antizyme 3 (OAZ3) were potential GGN1 interaction partners . We determined the regions mediating the interactions and further showed the interactions between the proteins in mammalian cells by colocalization and coimmunoprecipitation experiments . Our work suggested that GGN1, GGNBP1, GGNBP2 and OAZ3 could be involved in a common process associated with spermatogenesis. FEBS Lett, 2005 Jan 17, 579(2), 512 - 6 A role for the non-phosphorylated form of yeast Snf1: tolerance to toxic cations and activation of potassium transport; Portillo F et al.; The Snf1/AMP-activated protein kinases play a key role in stress responses of eukaryotic cells . In the yeast Saccharomyces cerevisiae Snf1 is regulated by glucose depletion, which triggers its phosphorylation at Thr210 and concomitant increase in activity . Activated yeast Snf1 is required for the metabolic changes allowing starvation tolerance and utilization of alternative carbon sources . We now report a function for the non-activated form of Snf1: the regulation of the Trk high-affinity potassium transporter, encoded by the TRK1 and TRK2 genes . A snf1Delta strain is hypersensitive in high-glucose medium to different toxic cations, suggesting a hyperpolarization of the plasma membrane driving increased cation uptake . This phenotype is suppressed by the TRK1 and HAL5 genes in high-copy number consistent with a defect in K(+) uptake mediated by the Trk system . Accordingly, Rb(+) uptake and intracellular K(+) measurements indicate that snf1Delta is unable to fully activate K(+) import . Genetic analysis suggests that the weak kinase activity of the non-phosphorylated form of Snf1 activates Trk in glucose-metabolizing yeast cells . The effect of Snf1 on Trk is probably indirect and could be mediated by the Sip4 transcriptional activator. FEBS Lett, 2005 Jan 17, 579(2), 507 - 11 Identification of a novel member of yeast mitochondrial Hsp70-associated motor and chaperone proteins that facilitates protein translocation across the inner membrane; Yamamoto H et al.; Here, we report the identification of yeast 15-kD Tim15/Zim17, a new member of mitochondrial Hsp70 (mtHsp70)-associated motor and chaperone (MMC) proteins . The 15-kD MMC protein is a peripheral inner membrane protein with a zinc-finger motif . Depletion of the 15-kD protein led to impaired import of presequence-containing proteins into the matrix in vivo and in vitro . Overexpression of the 15-kD protein rescued the functional defects of mtHsp70 in ssc1-3 cells, and a fusion protein containing the 15-kD protein physically interacts with purified mtHsp70 . Tim15/Zim17 therefore cooperates with mtHsp70 to facilitate import of presequence-containing proteins into the matrix. Ai Zheng, 2005 Jan, 24(1), 40 - 6 {Screening ubiquitin/ribosomal protein s27a by yeast two-hybridization system.}; Huang GH et al.; BACKGROUND & OBJECTIVE: Protein kinase CK2, a kind of ubiquitous eukaryotic messenger-independent protein serine/threonine kinase, plays a vital role in cell differentiation and proliferation, signal transduction and procession . Activity of CK2 in hematopoietic cells is 2-8 folds higher than that in relevant normal tissues, moreover changes of CK2 activity are correlated to tumor growth . In order to investigate the mechanism of its effect on hematopoietic cells, we used yeast two-hybridization screening the proteins interacting with protein kinase CK2alpha' subunit from HL-60 cells cDNA library . METHODS: The target CK2alpha' cDNA was obtained by amplifying recombinant plasmid pTHCK2A', containing human protein kinase CK2alpha' subunit cDNA, through polymerase chain reaction (PCR) . Pst I/Nde I-digested PCR products were directionally cloned into DNA-BD vector pGBKT7, which had also been digested by Pst I/Nde I . The recombinant plasmid was named yeast two-hybridization BD bait plasmid, and confirmed by DNA sequencing, and auto-activated experiments . Total RNA of HL-60 cells was extracted, CLONTECH switching mechanism at 5' end of RNA transcirpt method was used to construct a cDNA library in yeast cells . Library plasmid was named AD plasmid . BD plasmid and AD plasmid were co-transformed into competent yeast AH109 . Yeast two-hybridization was used to screen positive clones . RESULTS: Six proteins, interacting with human protein kinase CK2alpha' subunit, were screened . DNA sequencing and homology comparison showed that one of the proteins was highly homologous with ubiquitin/ribosomal protein S27a (99.8%) . CONCLUSION: Using yeast two-hybridization system could screen out ubiquitin/ribosomal protein S27a, which may interact with human protein kinase CK2alpha' subunit, from HL-60 cells. Oncogene, 2005 Jan 10, 24(2), 217 - 29 Yeast polo-like kinases: functionally conserved multitask mitotic regulators; Lee KS et al.; The polo-like kinases (Plks) are a conserved subfamily of Ser/Thr protein kinases that play pivotal roles in regulating various cellular and biochemical events at multiple stages of M phase . Genetic and biochemical data revealed that both the budding yeast and the fission yeast polo kinase homologs (Cdc5 and Plo1, respectively) bear remarkable functional similarities with those in metazoan organisms, suggesting that the role of Plks is largely conserved throughout evolution . Thus, studies on Plks in genetically amenable lower eucaryotic organisms may yield valuable insights into the function of Plks in higher eucaryotic organisms . In this review, common properties and distinct functions of Cdc5 and Plo1 will be discussed and compared to properties and functions of Plks in higher eucaryotic organisms. Appl Environ Microbiol, 2005 Jan, 71(1), 312 - 9 Isolation of Auxotrophic Mutants of Diploid Industrial Yeast Strains after UV Mutagenesis; Hashimoto S et al.; Auxotrophic mutants of the yeast Saccharomyces cerevisiae are usually isolated in haploid strains because the isolation of recessive mutations in diploids is thought to be difficult due to the presence of two sets of genes . We show here that auxotrophic mutants of diploid industrial sake yeast strains were routinely obtained by a standard mutant selection procedure following UV mutagenesis . We isolated His(-), Met(-), Lys(-), Trp(-), Leu(-), Arg(-), and Ura(-) auxotrophic mutants of five sake strains, Kyokai no . 7, no . 9, no . 10, no . 701, and no . 901, by screening only 1,700 to 3,400 colonies from each treated strain . Wild-type alleles were cloned and used as markers for transformation . With HIS3 as a selectable marker, the yeast TDH3 overexpression promoter was inserted upstream of ATF1, encoding alcohol acetyltransferase, by one-step gene replacement in a his3 mutant of Kyokai no . 7 . The resulting strain contained exclusively yeast DNA, making it acceptable for commercial use, and produced a larger amount of isoamyl acetate, a banana-like flavor . We argue that the generally recognized difficulty of isolating auxotrophic mutants of diploid industrial yeast strains is misleading and that genetic techniques used for haploid laboratory strains are applicable for this purpose. J Biol Chem . 2005 Jan 6; {Epub ahead of print} The binding mechanism of the yeast F1-ATPase inhibitory peptide . Role of catalytic intermediates and enzyme turnover; Corvest V et al.; The mechanism of inhibition of yeast mitochondrial F1-ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites . Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (beta-Tyr345Trp) . Fluorescence quenching of a beta-Trp345 indicates that the binding of MgADP to F1 can be described as 3 binding sites with dissociation constants of Kd1=10+/-2 nM, Kd2=0.22+/-0.03 microM, Kd3=16.3+/-0.2 microM . In addition, the ATPase activity of the beta-Trp345 enzyme followed simple Michaelis-Menten kinetics with a corresponding Km of 55 microM . Values for the Kd for MgATP were estimated and indicate that the Km (55 microM) for ATP hydrolysis corresponds to filling the third catalytic site on F1 . IF1 binds very slowly to F1-ATPase depleted of nucleotides and under unisite conditions . The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 microM, but decreased thereafter . The rate of inhibition was half-maximal at 5 microM MgATP, which is 10 fold lower than the Km for ATPase . The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis . A model is proposed that suggests that IF1 binds rapidly, but loosely to F1 with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP. Lipids, 2004 Aug, 39(8), 747 - 52 A mutation in sphingolipid synthesis suppresses defects in yeast ergosterol metabolism; Valachovic M et al.; A mutation in an otherwise nonessential ERG2 gene is synthetically lethal when combined with mutations in two transcription factors encoded by the UPC2 and ECM22 genes . Employing UV mutagenesis, we isolated a suppressor of the triple mutant erg2delta upc2delta ecm22delta . The morpholine-resistant phenotype of the suppressor was used to identify the suppressor as a mutation in the ELO3 gene . In an expression study on tridemorph-containing medium, using the inducible GAL1 promoter fused to the ELO3 open reading frame, we demonstrated that suppression occurred only when ELO3 was not expressed . ELO3 encodes an enzyme involved in sphingolipid synthesis required for long-chain FA synthesis . Surprisingly, a deletion of ELO2, also required for the synthesis of sphingolipid-containing long-chain FA, did not suppress the erg2delta upc2delta ecm22delta triple mutant . The sterol composition of the upc2delta ecm22delta double mutant reflected regulation of the latter part of the ergosterol synthesis by the Upc2p and Ecm22p transcription factors . This study demonstrates a synergistic relationship between two lipid species, sterols and sphingolipids. J Biol Chem . 2005 Jan 6; {Epub ahead of print} Synthase III-dependent chitin is bound to different acceptors depending on location on the cell wall of budding yeast; Cabib E et al.; In yeast, chitin is laid down at three locations: a ring at the mother-bud neck, the primary septum and, after cytokinesis, the cell wall of the daughter cell . Some of the chitin is free, the remainder attached to beta(1-3)glucan or beta(1-6)glucan . We recently reported that the chitin ring contributes to the prevention of growth at the mother-bud neck and hypothesized that this inhibition is achieved by a preferential binding of chitin to beta(1-3)glucan at that site . Here, we devised a novel strategy for the analysis of chitin cross-links in 14C-glucosamine-labeled cell walls, involving solubilization in water of alkali-treated walls by carboxymethylation . Intact cell walls or their digestion products with beta(1-3)glucanase or beta(1-6)glucanase were carboxymethylated and fractionated on size columns, and the percentage of chitin bound to different polysaccharides was calculated . Chitin dispersed in the wall was labeled in maturing unbudded cells and that of the ring in early budding cells . The former was mostly attached to beta(1-6)glucan and the latter to beta(1-3)glucan . This confirmed our hypothesis and indicated that the cell has mechanisms to attach chitin, a water-insoluble substance, synthesized here through chitin synthase III, to different acceptors, depending on location . In contrast, most of the chitin synthase II-dependent chitin of the primary septum was free, with the remainder linked to beta(1-3)glucan. J Biol Chem . 2005 Jan 6; {Epub ahead of print} Extended DNA binding site in Pot1 broadens sequence specificity to allow recognition of heterogeneous fission yeast telomeres; Trujillo KM et al.; The Pot1 (protection of telomeres) protein binds to single-stranded telomeric DNA and is essential for the protection of chromosome ends from degradation and end-to-end fusions . The Pot1 amino-terminal DNA-binding domain, Pot1N, adopts an oligonucleotide/oligosaccharide-binding fold and binds GGTTAC motifs cooperatively and with exceptionally high sequence specificity . We have now examined DNA-binding to naturally occurring telomeric substrates based on the analysis of 100 cloned chromosome ends and in the context of the full-length Pot1 protein . Here, we describe several important differences between Pot1 and Pot1N with apparent consequences for chromosome end protection . Specifically, full length Pot1-DNA complexes are more stable and the minimal binding site for a Pot1 monomer is extended into two adjacent telomeric repeats . We provide evidence that Pot1 contains a second DNA-binding motif that recognizes DNA with reduced sequence specificity compared to the domain present in Pot1N . The two DNA-binding motifs cooperate, whereby the amino-terminal OB-fold determines the registry of binding and the internal DNA-binding motif stabilizes the complex and expands the protected region towards the 3' end . Consistent with a role in chromosome end capping, Pot1 prevents access of telomerase to the 3' end and protects against exonucleolytic degradation. Mol Biol Cell . 2005 Jan 5; {Epub ahead of print} The Yeast Endosomal Na+(K+)/H+ Exchanger Nhx1 Regulates Cellular pH to Control Vesicle Trafficking; Brett CL et al.; Monitoring Editor: Sandra Schmid The relationship between endosomal pH and function is well documented in viral entry, endosomal maturation, receptor recycling, and vesicle targeting within the endocytic pathway . However, specific molecular mechanisms that either sense or regulate luminal pH to mediate these processes have not been identified . Herein we describe the use of novel, compartment-specific pH indicators to demonstrate that yeast Nhx1, an endosomal member of the ubiquitous NHE family of Na(+)/H(+) exchangers, regulates luminal and cytoplasmic pH to control vesicle trafficking out of the endosome . Loss of Nhx1 confers growth sensitivity to low pH stress, and concomitant acidification and trafficking defects, which can be alleviated by weak bases . Conversely, weak acids cause wild-type yeast to present nhx1Delta trafficking phenotypes . Finally, we report that Nhx1 transports K(+) in addition to Na(+), suggesting that a single mechanism may responsible for both pH and K(+)-dependent endosomal processes . This presents the newly defined family of eukaryotic endosomal NHE as novel targets for pharmacological inhibition to alleviate pathological states associated with organellar alkalinization. J Am Anim Hosp Assoc, 2005 Jan-Feb, 41(1), 12 - 21 Efficacy of boric-complexed zinc and acetic-complexed zinc otic preparations for canine yeast otitis externa; Mendelsohn CL et al.; The purpose of this 2-week, double-blinded, controlled clinical trial was to evaluate the efficacy of topical amino acid-complexed zinc gluconate formulated with boric acid (ZGB) or acetic acid (ZGA) versus a topical placebo in the treatment of yeast otitis externa in dogs . Included in the study were dogs with otitis externa and a cytopathological finding of yeast organisms in the affected ear . Ears were treated with the placebo, ZGA, or ZGB medications . Yeast counts as well as clinical appearance of the ears were monitored . Results revealed that ZGB significantly reduced the number of yeast organisms in cases of otitis externa. Cell Stress Chaperones, 2004 Winter, 9(4), 359 - 68 Investigating the protein-protein interactions of the yeast Hsp90 chaperone system by two-hybrid analysis: potential uses and limitations of this approach; Millson SH et al.; The Hsp90 chaperone cycle involves sequential assembly of different Hsp90-containing multiprotein complexes, the accessory proteins ("cochaperones") that are associated with these complexes being exchanged as the cycle proceeds from its early to its late stages . To gain insight as to whether the 2-hybrid system could be used to probe the interactions of this Hsp90 system, yeast transformants were constructed that express the Gal4p deoxyribonucleic acid-binding domain (BD) fused to the 2 Hsp90 isoforms and the various Hsp90 system cochaperones of yeast . These "bait" fusions were then introduced by mating into other transformants expressing nearly all the 6000 proteins of yeast expressed as fusions to the Gal4p activation domain (AD) . High throughput 2-hybrid screening revealed the ability of Hsp90 and Hsp90 system cochaperones to engage in stable interactions in vivo, both with each other and with the various other proteins of the yeast proteome . Consistent with the transience of most chaperone associations, interactions to Hsp90 itself were invariably weak and generally influenced by stress . Mutations within a Hsp90-BD bait fusion and an AD-Cdc37 "prey" fusion were used to provide in vivo confirmation of the in vitro data that shows that Cdc37p is interacting with the "relaxed" conformation of Hsp90 and also to provide indications that Cdc37p needs to be phosphorylated at its N-terminus for any appreciable interaction with Hsp90 . A number of potentially novel cochaperone interactions were also identified, providing a framework for these to be analyzed further using other techniques. Am J Pathol, 2005 Jan, 166(1), 265 - 74 A human yeast artificial chromosome containing the multiple endocrine neoplasia type 2B Ret mutation does not induce medullary thyroid carcinoma but does support the growth of kidneys and partially rescues enteric nervous system development in Ret-deficient mice; Skinner MA et al.; We generated a line of transgenic mice using a yeast artificial chromosome containing the Ret mutation responsible for the multiple endocrine neoplasia type 2B syndrome (MEN 2B) . The resulting animals did not develop any of the expected neoplasms associated with MEN 2B . Transgenic animals were then bred with animals lacking murine Ret (Ret(M)) to further evaluate the function of human mutated Ret (Ret(H)(2B)) in the murine context . Whereas mice lacking Ret(M) exhibit intestinal aganglionosis and the absence of kidneys with other genitourinary anomalies, expression of the Ret(H)(2B) transgene in Ret(M)-deficient mice allowed significant renal development with a partial rescue of the enteric nervous system . These Ret(H)(2B)-positive/Ret(M)-deficient mice exhibit normal Ret expression and survive longer than Ret(M)-deficient mice, but still die at 3 to 5 days of age with evidence of enterocolitis . We conclude that the normal expression of a human Ret proto-oncogene with the MEN 2B mutation does not cause any features of MEN 2B in mice . Although the gene is normally expressed in the appropriate target tissues, there is incomplete phenotypic rescue in mice lacking murine Ret . These results suggest important interspecies differences between humans and mice in the function of the Ret oncogene. J Cell Biol, 2005 Jan 3, 168(1), 103 - 15 Mdm31 and Mdm32 are inner membrane proteins required for maintenance of mitochondrial shape and stability of mitochondrial DNA nucleoids in yeast; Dimmer KS et al.; The MDM31 and MDM32 genes are required for normal distribution and morphology of mitochondria in the yeast Saccharomyces cerevisiae . They encode two related proteins located in distinct protein complexes in the mitochondrial inner membrane . Cells lacking Mdm31 and Mdm32 harbor giant spherical mitochondria with highly aberrant internal structure . Mitochondrial DNA (mtDNA) is instable in the mutants, mtDNA nucleoids are disorganized, and their association with Mmm1-containing complexes in the outer membrane is abolished . Mutant mitochondria are largely immotile, resulting in a mitochondrial inheritance defect . Deletion of either one of the MDM31 and MDM32 genes is synthetically lethal with deletion of either one of the MMM1, MMM2, MDM10, and MDM12 genes, which encode outer membrane proteins involved in mitochondrial morphogenesis and mtDNA inheritance . We propose that Mdm31 and Mdm32 cooperate with Mmm1, Mmm2, Mdm10, and Mdm12 in maintenance of mitochondrial morphology and mtDNA. J Am Chem Soc, 2005 Jan 12, 127(1), 92 - 9 EPR and optical spectroscopic studies of Met80X mutants of yeast ferricytochrome c . Models for intermediates in the alkaline transition; Silkstone GG et al.; The ferric forms of Met80X mutants of yeast iso-1-cytochrome c (X = Ala, Ser, Asp, and Glu) display EPR and optical spectra that are strongly pH dependent . At low pH values (pH approximately 5) the sixth coordination sites are filled by H(2)O that, on elevating the pH, is replaced by OH(-) in the cases of Met80Ala and -Ser (pK approximately 5.6 and 5.9, respectively) and by a lysine amino group in the cases of Met80Asp and -Glu (pK approximately 9.3 and 11.6, respectively) . The ligand sets and the pK values of the transitions are rationalized in terms of the structure of the heme pocket, and a possible mechanism of the "trigger" in the alkaline transition of the native protein is suggested. Genes Dev, 2005 Jan 1, 19(1), 90 - 103 Yeast poly(A)-binding protein, Pab1, and PAN, a poly(A) nuclease complex recruited by Pab1, connect mRNA biogenesis to export; Dunn EF et al.; In eukaryotic cells, pre-mRNAs undergo extensive processing in the nucleus prior to export . Processing is subject to a quality-control mechanism that retains improperly processed transcripts at or near sites of transcription . A poly(A) tail added by the normal 3'-processing machinery is necessary but not sufficient for export . Retention depends on the exosome . In this study, we identify the poly(A)-binding protein, Pab1, and the poly(A) nuclease, PAN, as important factors that couple 3' processing to export . Pab1 contains a nonessential leucine-rich nuclear export signal and shuttles between the nucleus and the cytoplasm . It can exit the nucleus either as cargo of exportin 1 or bound to mRNA . Pab1 is essential but several bypass suppressors have been identified . Deletion of PAB1 from these bypass suppressor strains results in exosome-dependent retention at sites of transcription . Retention is also seen in cells lacking PAN, which Pab1 is thought to recruit and which may be responsible for the final step of mRNA biogenesis, trimming of the poly(A) tail to the length found on newly exported mRNAs . The studies presented here suggest that proper loading of Pab1 onto mRNAs and final trimming of the tail allows release from transcription sites and couples pre-mRNA processing to export. Mol Cell, 2005 Jan 7, 17(1), 49 - 59 Inactivation of the Cdc25 Phosphatase by the Stress-Activated Srk1 Kinase in Fission Yeast; Lopez-Aviles S et al.; The mechanisms by which environmental stress regulates cell cycle progression are poorly understood . In fission yeast, we show that Srk1 kinase, which associates with the stress-activated p38/Sty1 MAP kinase, regulates the onset of mitosis by inhibiting the Cdc25 phosphatase . Srk1 is periodically active in G2, and its overexpression causes cell cycle arrest in late G2 phase, whereas cells lacking srk1 enter mitosis prematurely . We find that Srk1 interacts with and phosphorylates Cdc25 at the same sites phosphorylated by the Chk1 and Cds1 (Chk2) kinases and that this phosphorylation is necessary for Srk1 to delay mitotic entry . Phosphorylation by Srk1 causes Cdc25 to bind to Rad24, a 14-3-3 protein family member, and accumulation of Cdc25 in the cytoplasm . However, Srk1 does not regulate Cdc25 in response to replication arrest or DNA damage but, rather, during a normal cell cycle and in response to nongenotoxic environmental stress. J Struct Biol, 2005 Jan, 149(1), 111 - 5 Gene silencing pathway RNA-dependent RNA polymerase of Neurospora crassa: yeast expression and crystallization of selenomethionated QDE-1 protein; Laurila MR et al.; The RNA-dependent RNA polymerase, QDE-1, is a component of the RNA silencing pathway in Neurospora crassa . The enzymatically active carboxy-terminal fragment QDE-1 DeltaN has been expressed in Saccharomyces cerevisiae in the presence and absence of selenomethionine (SeMet) . The level of SeMet incorporation was estimated by mass spectrometry to be approximately 98% . Both native and SeMet proteins were crystallized in space group P2(1) with unit cell parameters a=101.2, b=122.5, c=114.4A, beta=108.9 degrees , and 2 molecules per asymmetric unit . The native and SeMet crystals diffract to 2.3 and 3.2A, respectively, the latter are suitable for MAD structure determination. Biochem Biophys Res Commun, 2005 Feb 4, 327(1), 143 - 9 Identification of an evolutionary conserved SURF-6 domain in a family of nucleolar proteins extending from human to yeast; Polzikov M et al.; The mammalian SURF-6 protein is localized in the nucleolus, yet its function remains elusive in the recently characterized nucleolar proteome . We discovered by searching the Protein families database that a unique evolutionary conserved SURF-6 domain is present in the carboxy-terminal of a novel family of eukaryotic proteins extending from human to yeast . By using the enhanced green fluorescent protein as a fusion protein marker in mammalian cells, we show that proteins from distantly related taxonomic groups containing the SURF-6 domain are localized in the nucleolus . Deletion sequence analysis shows that multiple regions of the SURF-6 protein are capable of nucleolar targeting independently of the evolutionary conserved domain . We identified that the Saccharomyces cerevisiae member of the SURF-6 family, named rrp14 or ykl082c, has been categorized in yeast databases to interact with proteins involved in ribosomal biogenesis and cell polarity . These results classify SURF-6 as a new family of nucleolar proteins in the eukaryotic kingdom and point out that SURF-6 has a distinct domain within the known nucleolar proteome that may mediate complex protein-protein interactions for analogous processes between yeast and mammalian cells. Biochem Biophys Res Commun, 2005 Feb 11, 327(2), 597 - 603 Random mutagenesis of the B'A' core domain of yeast DNA topoisomerase II and large-scale screens of mutants resistant to the anticancer drug etoposide; Jiang X; Mutagenic PCR method was applied to introduce point mutations to the B'A' core domain of yeast DNA topoisomerase II . Screens for mutants resistant to the anticancer drug etoposide were carried out in a yeast ts system in the presence of high concentrations of the drug or in a drug-hypersensitive genetic background . 129 mutants were obtained from a total of 47,000 transformants . Nucleotide sequencing of 40 selected mutants showed that a large number of the mutations map to regions encoding the linker that joins the ATPase domain to the B' module and the B'A' linker . Significant reduction in catalytic activity was evident for a large fraction of mutant enzymes and all mutants were also resistant to amsacrine, another topoisomerase II drug with a different chemical structure, suggesting that few of the mutations reflect simple changes of specific amino acid side chains that are directly involved in enzyme-drug interactions. Biochemistry, 2005 Jan 11, 44(1), 377 - 86 Kinetic Analysis of YPD1-Dependent Phosphotransfer Reactions in the Yeast Osmoregulatory Phosphorelay System; Janiak-Spens F et al.; In Saccharomyces cerevisiae, the histidine-containing phosphotransfer (HPt) protein YPD1 transfers phosphoryl groups between the three different response regulator domains of SLN1, SSK1, and SKN7 (designated R1, R2, and R3, respectively) . Together these proteins form a branched histidine-aspartic acid phosphorelay system through which cells can respond to hyperosmotic and other environmental stresses . The in vivo order of phosphotransfer reactions is believed to proceed from SLN1-R1 to YPD1 and then subsequently to SSK1-R2 or SKN7-R3 . The individual phosphoryl transfer reactions between YPD1 and the response regulator domains have been examined kinetically . A maximum forward rate constant of 29 s(-)(1) was determined for the reaction between SLN1-R1 approximately P and YPD1 with a K(d) of 1.4 muM for the SLN1-R1 approximately P.YPD1 complex . In the subsequent reactions, phosphotransfer from YPD1 to SSK1-R2 is very rapid (160 s(-)(1)) and is strongly favored over phosphotransfer to SKN7-R3 . Phosphotransfer reactions between YPD1 and SLN1-R1 or SKN7-R3 were reversible . In contrast, no reverse transfer from SSK1-R2 approximately P to YPD1 was observed . These findings are consistent with the notion that SSK1 is constitutively phosphorylated under normal osmotic conditions . In addition, we have examined the roles of several conserved amino acid residues surrounding the phosphorylatable histidine (H64) of YPD1 using phosphoryl transfer reactions involving YPD1 mutants . With respect to phosphoryl transfer from SLN1-R1 approximately P, only one YPD1 mutant (K67A) exhibited an increase in K(d) and thus affects binding of YPD1 to SLN1-R1 approximately P, whereas other mutants (R90A, Q86A, and G68Q) showed a decrease in phosphoryl transfer rate . Only the G68Q-YPD1 mutant was significantly affected in phosphotransfer to SSK1-R2 ( approximately 680-fold decrease in rate in comparison to wild-type) . This is the first report of a kinetic analysis of a eukaryotic "two-component" histidine-aspartic acid phosphotransfer system, enabling a comparison of the transfer rates and binding constants to the few bacterial systems that have been studied this way. Proteomics . 2005 Jan 3; {Epub ahead of print} Peeling the yeast protein network; Wuchty S et al.; A set of highly connected proteins (or hubs) plays an important role for the integrity of the protein interaction network of Saccharomyces cerevisae by connecting the network's intrinsic modules {1, 2} . The importance of the hubs' central placement is further confirmed by their propensity to be lethal . However, although highly emphasized, little is known about the topological coherence among the hubs . Applying a core decomposition method which allows us to identify the inherent layer structure of the protein interaction network, we find that the probability of nodes both being essential and evolutionary conserved successively increases toward the innermost cores . While connectivity alone is often not a sufficient criterion to assess a protein's functional, evolutionary and topological relevance, we classify nodes as globally and locally central depending on their appearance in the inner or outer cores . The observation that globally central proteins participate in a substantial number of protein complexes which display an elevated degree of evolutionary conservation allows us to hypothesize that globally central proteins serve as the evolutionary backbone of the proteome . Even though protein interaction data are extensively flawed, we find that our results are very robust against inaccurately determined protein interactions. Biochemistry (Mosc), 2004 Dec, 69(12), 1391 - 8 Regulation of NAD(+)-Dependent Isocitrate Dehydrogenase in the Citrate Producing Yeast Yarrowia lipolytica; Morgunov IG et al.; The mechanism of the increased accumulation (overproduction) of citric acids in the yeast Yarrowia lipolytica while growing in the presence of glucose under nitrogen deficiency was investigated . The limitation of the yeast growth by the source of nitrogen decreases the total content of nucleotides and increases the ratios of ATP/AMP and NADH/NAD+ . NAD+-Dependent isocitrate dehydrogenase, an enzyme of the tricarboxylic acid cycle playing a key role in the regulation of biosynthesis of citric and isocitric acids, was isolated from Y . lipolytica . The molecular weights of the native enzyme and its subunits were found to be 412 and 52 kD, respectively . It is concluded that the enzyme is a homooligomer consisting of eight subunits . Investigation of the effect of some intermediates of the tricarboxylic acid cycle on the activity of this enzyme suggests that the enhanced excretion of citric acids can be caused by the inhibition of NAD+-dependent isocitrate dehydrogenase due to the decrease in the content of AMP and increase in the NADH/NAD+ ratio in the cells of Y . lipolytica under depletion of nitrogen. Biochemistry (Mosc), 2004 Nov, 69(11), 1310 - 7 A New Alkalitolerant Yarrowia lipolytica Yeast Strain Is a Promising Model for Dissecting Properties and Regulation of Na(+)-Dependent Phosphate Transport Systems; Zvyagilskaya RA et al.; A newly isolated osmo-, salt-, and alkalitolerant Yarrowia lipolytica yeast strain is distinguished from other yeast species by its capacity to grow vigorously at alkaline pH values (9.7), which makes it a promising model organism for studying Na+-dependent phosphate transport systems in yeasts . Phosphate uptake by Y . lipolytica cells grown at pH 9.7 was mediated by several kinetically discrete Na+-dependent systems specifically activated by Na+ . One of these, a low-affinity transporter, operated at high concentrations of extracellular phosphate . The other two, high-affinity systems, maximally active in phosphate-starved cells, were repressed or derepressed depending on the prevailing extracellular phosphate concentration and pH value . The contribution of Na+/P(i)-cotransport systems to the total cellular phosphate uptake progressively increased with increasing pH, reaching its maximum at pH >/= 9. Pest Manag Sci . 2004 Dec 31; {Epub ahead of print} Expression of a wheat cytochrome P450 monooxygenase in yeast and its inhibition by glyphosate; Xiang WS et al.; Glyphosate is a non-selective herbicide which acts by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase . Wheat cytochrome P450 monooxygenase specifically catalyzes the metabolism of some sulfonylurea herbicides such as chlorsulfuron and triasulfuron . Here we report that glyphosate is an inhibitor of a wheat cytochrome (CYP71C6v1), the cDNA of which was amplified by RT-PCR and heterologously expressed in yeast . The microsomal fractions derived from this strain had a Soret peak at 502 nm in the reduced carbon monoxide difference spectrum, which is a typical spectral characteristic . The addition of glyphosate to the microsomal protein resulted in a Type II spectrum indicative of binding via the nitrogen group to haem of cytochrome P450 as a sixth ligand . A spectral dissociation constant, K(s) of 70 micromol litre(-1) was observed and an IC(50) of 11 micromol litre(-1) was found for glyphosate inhibition of CYP71C6v1 P450 activity . Copyright (c) 2005 Society of Chemical Industry. Anal Chem, 2005 Jan 1, 77(1), 344 - 9 Determination of methionine and selenomethionine in selenium-enriched yeast by species-specific isotope dilution with liquid chromatography-mass spectrometry and inductively coupled plasma mass spectrometry detection; McSheehy S et al.; Selenomethionine (SeMet) and methionine (Met), liberated by acid hydrolysis of selenium-enriched yeast, were quantified by liquid chromatography-mass spectrometry (LC/MS) using standard additions calibrations as well as isotope dilution (ID) based on species-specific (13)C-enriched spikes . LC inductively coupled plasma mass spectrometry (ICPMS) was also employed for the quantification of SeMet, and (74)Se-enriched SeMet was used for ID calibration . The results were evaluated to ascertain the feasibility of using these methods in a campaign to certify selenized yeast . Good agreement was found between the methods, which, when averaged, gave concentrations of 5482.2 +/- 101 and 3256.9 +/- 217.4 microg/g for Met and SeMet, respectively . This corresponds to a 1.68:1 Met-to-SeMet ratio in the yeast . Quantification by ID LC/MS and LC ICPMS yields the most precise sets of results with relative standard deviations in the range 0.5-1.3% (n = 6) . A total selenium concentration of 2064.6 +/- 45.4 microg/g was obtained for this yeast material . The extraction efficiency and a mass balance budget were determined . Acid hydrolysis liberated 81.0% of the total selenium present . SeMet comprised 79.0% of the extracted selenium and 63.9% of the total selenium present in the yeast. Sci China C Life Sci, 2004 Oct, 47(5), 442 - 8 Androgen receptor coregulator ARA267-alpha interacts with death receptor-6 revealed by the yeast two-hybrid; Mai T et al.; ARA267-alpha is a newly identified androgen receptor coactivator . In order to further elucidate its precise role in cells, using the ARA267-alpha fragment containing four PHD and one SET conserved domains as bait we revealed an ARA267-alpha-PHD-SET-interacting protein, death receptor-6 (DR6), in the yeast two-hybrid screening . DR6 is the member of TNF receptor family and has a death domain in its intracellular cytoplasmic portion (DR6cp) to mediate the cell apoptosis . The interaction between ARA267-alpha-PHD-SET and DR6cp was confirmed in vitro and in vivo . Our finding implied that androgen signaling pathway might cross talk with apoptosis signaling pathway through the interaction between ARA267-alpha and DR6. Free Radic Res, 2004 Sep, 38(9), 1019 - 24 Ascorbate restores lifespan of superoxide-dismutase deficient yeast; Krzepilko A et al.; Yeast (Saccharomyces cerevisiae) mutants lacking CuZn-superoxide dismutase (CuZnSOD) are hypersensitive to oxygen and have significantly decreased replicative life span . Both these defects can be ameliorated by exogenous ascorbate . The effect of ascorbate on life span is complicated by auto-oxidation of its compound in the medium . If negative effects of auto-oxidation are prevented by exchange of the medium, ascorbate prolongs not only mean but also maximal replicative life span of the yeast in the atmosphere of air and of pure oxygen . These results demonstrate that life span shortening due to the lack of a vital antioxidant enzyme can be ameliorated by a low-molecular weight antioxidant. Free Radic Res, 2004 Nov, 38(11), 1159 - 65 Limited effectiveness of antioxidants in the protection of yeast defective in antioxidant proteins; Lewinska A et al.; Efficacy of several antioxidants in the protection of the yeast Saccharomyces cerevisiae mutants deficient in CuZnSOD and deficient in glutaredoxin 5 to growth restriction induced by oxidants was studied . Ascorbate and glutathione protected the Deltasod1 and Deltagrx5 mutants against the effects of t-butyl hydroperoxide and cumene hydroperoxide, Deltasod1 mutants against oxytetracycline and Deltagrx5 mutants against menadione and 2,2'-azobis-(2-amidinopropane) . However, Tempol, Trolox and melatonin were much less effective, showing prooxidative effects and, at high concentrations, hampering the growth of the mutants in the absence of exogenous oxidants . These results point to a complication of cellular effects of antioxidants by their prooxidative effects and to the usefulness of cellular tests to evaluate the biological effectiveness of antioxidants. Mech Ageing Dev, 2005 Feb, 126(2), 243 - 8 Yeast longevity and aging-the mitochondrial connection; Jazwinski SM; Studies of the yeast Saccharomyces cerevisiae reveal four processes determining life span: metabolism, stress resistance, chromatin-dependent gene regulation, and genome stability . The retrograde response, which signals mitochondrial dysfunction resulting in changes in nuclear gene expression, extends yeast life span and is induced during normal aging . This response involves extensive metabolic adaptations . The retrograde response links metabolism and genome stability during yeast aging . A reduction in the availability of nutrients also extends yeast life span . This metabolic mechanism operates by pathways distinct from the retrograde response, although it shares with the latter some longevity effectors . Life extension by calorie restriction entails re-modeling of mitochondrial function . The retrograde response appears to compensate for age changes, while calorie restriction may be a preventive mechanism . The maintenance of age asymmetry between the mother and daughter yeast cells also depends on mitochondrial function . Loss of this age asymmetry occurs during normal yeast aging and may be a paradigm for stem cell aging . The importance of mitochondrial integrity in yeast longevity is emphasized by the role of prohibition function in attenuating oxidative damage . Our studies point to the central role of mitochondria in yeast aging . They highlight the importance of the maintenance of mitochondrial membrane potential, which drives the transport of biosynthetic precursors derived from the Krebs cycle . Common threads weave their way through the studies of aging in yeast and in other model organisms . This suggests conserved features of aging across phyla. Curr Biol, 2004 Dec 29, 14(24), 2183 - 96 Spo13 facilitates monopolin recruitment to kinetochores and regulates maintenance of centromeric cohesion during yeast meiosis; Katis VL et al.; BACKGROUND: Cells undergoing meiosis perform two consecutive divisions after a single round of DNA replication . During the first meiotic division, homologous chromosomes segregate to opposite poles . This is achieved by (1) the pairing of maternal and paternal chromosomes via recombination producing chiasmata, (2) coorientation of homologous chromosomes such that sister chromatids attach to the same spindle pole, and (3) resolution of chiasmata by proteolytic cleavage by separase of the meiotic-specific cohesin Rec8 along chromosome arms . Crucially, cohesin at centromeres is retained to allow sister centromeres to biorient at the second division . Little is known about how these meiosis I-specific events are regulated . RESULTS: Here, we show that Spo13, a centromere-associated protein produced exclusively during meiosis I, is required to prevent sister kinetochore biorientation by facilitating the recruitment of the monopolin complex to kinetochores . Spo13 is also required for the reaccumulation of securin, the persistence of centromeric cohesin during meiosis II, and the maintenance of a metaphase I arrest induced by downregulation of the APC/C activator CDC20 . CONCLUSION: Spo13 is a key regulator of several meiosis I events . The presence of Spo13 at centromere-surrounding regions is consistent with the notion that it plays a direct role in both monopolin recruitment to centromeres during meiosis I and maintenance of centromeric cohesion between the meiotic divisions . Spo13 may also limit separase activity after the first division by ensuring securin reaccumulation and, in doing so, preventing precocious removal from chromatin of centromeric cohesin. Biochim Biophys Acta, 2005 Jan 7, 1706(1-2), 117 - 25 The yeast cAMP protein kinase Tpk3p is involved in the regulation of mitochondrial enzymatic content during growth; Chevtzoff C et al.; During aerobic cell growth, mitochondria must meet energy demand either by adjusting cellular mitochondrial content or by adjusting ATP production flux, allowing a constant growth yield . On respiratory substrate, the Ras/cAMP pathway has been shown to be involved in this process in the yeast Saccharomyces cerevisiae . We show that of the three cAMP protein kinase catalytic subunits, Tpk3p is the one specifically involved in the regulation of cellular mitochondrial content when energy demand decreases . In decreased energy demand, the Deltatpk3 mitochondrial enzymatic content decreases leading to a subsequent decrease in the cellular growth rate . Moreover, enzymatic content decreases in the Deltatpk3 isolated mitochondria, suggesting that the amount of cellular mitochondria is not affected, but rather that the mitochondria are modified . Our study points to an important decrease in the cytochrome c content in the Deltatpk3 mitochondria, which leads to a decrease in the slipping process at the level of cytochrome-c-oxidase. Cell, 2004 Dec 29, 119(7), 991 - 1000 Pheromone-dependent destruction of the Tec1 transcription factor is required for MAP kinase signaling specificity in yeast; Bao MZ et al.; The yeast MAPK pathways required for mating versus filamentous growth share multiple components yet specify distinct programs . The mating-specific MAPK, Fus3, prevents crosstalk between the two pathways by unknown mechanisms . Here we show that pheromone signaling induces Fus3-dependent degradation of Tec1, the transcription factor specific to the filamentation pathway . Degradation requires Fus3 kinase activity and a MAPK phosphorylation site in Tec1 at threonine 273 . Fus3 associates with Tec1 in unstimulated cells, and active Fus3 phosphorylates Tec1 on T273 in vitro . Destruction of Tec1 requires the F box protein Dia2 (Digs-into-agar-2), and Cdc53, the Cullin of SCF (Skp1-Cdc53-F box) ubiquitin ligases . Notably, mutation of the phosphoacceptor site in Tec1, deletion of FUS3, or deletion of DIA2 results in a loss of signaling specificity such that pheromone pathway signaling erroneously activates filamentation pathway gene expression and invasive growth . Signal-induced destruction of a transcription factor for a competing pathway provides a mechanism for signaling specificity. Cell, 2004 Dec 29, 119(7), 981 - 90 Fus3-regulated Tec1 degradation through SCFCdc4 determines MAPK signaling specificity during mating in yeast; Chou S et al.; Signaling specificity is fundamental for parallel mitogen-activated protein kinase (MAPK) cascades that control growth and differentiation in response to different stimuli . In Saccharomyces cerevisiae, components of the pheromone-responsive MAPK cascade activate Fus3 and Kss1 MAPKs to induce mating and Kss1 to promote filamentation . Active Fus3 is required to prevent the activation of the filamentation program during pheromone response . How Fus3 prevents the crossactivation is not clear . Here we show that Tec1, a cofactor of Ste12 for the expression of filamentation genes, is rapidly degraded during pheromone response . Fus3 but not Kss1 induces Tec1 ubiquination and degradation through the SCFCdc4 ubiquitin ligase . T273 in a predicted high-affinity Cdc4 binding motif is phosphorylated by Fus3 both in vitro and in vivo . Tec1T273V blocks Tec1 ubiquitination and degradation and allows the induction of filamentation genes in response to pheromone . Thus, Fus3 inhibits filamentous growth during mating by degrading Tec1. J Biol Chem . 2004 Dec 24; {Epub ahead of print} Physical and genetic interactions link the yeast protein Zds1p with mRNA nuclear export; Estruch F et al.; Eukaryotic gene expression requires the export of mRNA from nucleus to the cytoplasm . The DEAD box protein Dbp5p is an essential export factor conserved from yeast to man . A fraction of Dbp5p forms a complex with nucleoporins of the cytoplasmic filaments of the nuclear pore complex (NPC) . Gfd1p was identified originally as a multicopy suppressor of the rat8-2 ts allele of DBP5 . Here we report that Dbp5p and Gfd1p interact with Zds1p, a protein previously identified as a multicopy suppressor in several yeast genetic screens . Using the 2-hybrid system we show that Zds1p interacts in vivo with both Gfd1p and Dbp5p . In vitro binding experiments reveal that Gfd1p and Dbp5p bind directly to the C-terminal part of Zds1p . In addition, ZDS1 interacts genetically with mutant alleles of genes encoding key factors in mRNA export, including DBP5 and MEX67 . Furthermore, deletion of ZDS1 or of both ZDS1 and the closely-related ZDS2 exacerbates the poly(A)+ export defects shown by dbp5-2 and mex67-5 mutants . We propose that Zds1p associates with the complex formed by Dbp5p, Gfd1p, and nucleoporins at the cytosolic fibrils of the NPC and is required for optimal mRNA export. Drug Metab Pharmacokinet, 2002, 17(2), 117 - 24 Molecular Cloning of Monkey CYP2C43 cDNA and Expression in Yeast; Matsunaga T et al.; A cDNA clone designated as CYP2C43 was isolated from the rhesus monkey liver cDNA library . The first 16 amino acid residues at the N-terminal region of this cDNA product were identical with those of P450 CMLd which have been purified and characterized as S-mephenytoin 4'-hydroxylase in monkey liver . The respective nucleotide and deduced amino acid sequences of CYP2C43 were 83% and 77%, identical to those of monkey CYP2C20 . Antibody against CYP2C9 detected a protein in the microsomes of yeast transformed CYP2C43 expression plasmid . The specific content of recombinant CYP2C43 was 78.0 pmol/mg protein and the yield was 4.23 nmol/l of the culture . CYP2C43 was able to metabolize S-mephenytoin stereo-selectively . The activity for S-mephenytoin in the microsomes reconstituted with or without cytochrome b(5) was found to be 96.2 or 23.7 pmol/min/nmol P450, respectively . CYP2C43, however, did not show any oxidative activity for tolbutamide . These results indicate that CYP2C43 is the second identified member of the monkey CYP2C subfamily and a cDNA clone encoding P450 CMLd in monkey. Plant Physiol, 2005 Jan, 137(1), 308 - 16 Epub 2004 Dec 23. The Cytokinin Requirement for Cell Division in Cultured Nicotiana plumbaginifolia Cells Can Be Satisfied by Yeast Cdc25 Protein Tyrosine Phosphatase . Implications for Mechanisms of Cytokinin Response and Plant Development; Zhang K et al.; Cultured cells of Nicotiana plumbaginifolia, when deprived of exogenous cytokinin, arrest in G2 phase prior to mitosis and then contain cyclin-dependent protein kinase (CDK) that is inactive because phosphorylated on tyrosine (Tyr) . The action of cytokinin in stimulating the activation of CDK by removal of inhibitory phosphorylation from Tyr is not a secondary downstream consequence of other hormone actions but is the key primary effect of the hormone in its stimulation of cell proliferation, since cytokinin could be replaced by expression of cdc25, which encodes the main Cdc2 (CDK)-Tyr dephosphorylating enzyme of yeast (Saccharomyces cerevisiae) . The cdc25 gene, under control of a steroid-inducible promoter, induced a rise in cdc25 mRNA, accumulation of p67(Cdc25) protein, and increase in Cdc25 phosphatase activity that was measured in vitro with Tyr-phosphorylated Cdc2 as substrate . Cdc25 phosphatase activity peaked during mitotic prophase at the time CDK activation was most rapid . Mitosis that was induced by cytokinin also involved increase in endogenous plant CDK Tyr phosphatase activity during prophase, therefore indicating that this is a normal part of plant mitosis . These results suggest a biochemical mechanism for several previously described transgene phenotypes in whole plants and suggest that a primary signal from cytokinin leading to progression through mitosis is the activation of CDK by dephosphorylation of Tyr. Proc Natl Acad Sci U S A, 2005 Jan 4, 102(1), 163 - 8 Epub 2004 Dec 23. The splicing of yeast mitochondrial group I and group II introns requires a DEAD-box protein with RNA chaperone function; Huang HR et al.; Group I and II introns self-splice in vitro, but require proteins for efficient splicing in vivo, to stabilize the catalytically active RNA structure . Recent studies showed that the splicing of some Neurospora crassa mitochondrial group I introns additionally requires a DEAD-box protein, CYT-19, which acts as an RNA chaperone to resolve nonnative structures formed during RNA folding . Here we show that, in Saccharomyces cerevisiae mitochondria, a related DEAD-box protein, Mss116p, is required for the efficient splicing of all group I and II introns, some RNA end-processing reactions, and translation of a subset of mRNAs, and that all these defects can be partially or completely suppressed by the expression of CYT-19 . Results for the aI2 group II intron indicate that Mss116p is needed after binding the intron-encoded maturase, likely for the disruption of stable but inactive RNA structures . Our results suggest that both group I and II introns are prone to kinetic traps in RNA folding in vivo and that the splicing of both types of introns may require DEAD-box proteins that function as RNA chaperones. J Biol Chem . 2004 Dec 23; {Epub ahead of print} Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids; Salnikova AB et al.; The {PSI+} prion determinant of Saccharomyces cerevisiae causes nonsense suppressor phenotype due to a reduced function of the translation termination factor Sup35 (eRF3) polymerized into amyloid fibrils . Prion state of the Rnq1 protein, {PIN+}, is required for the {PSI+} de novo generation, but not propagation . Yeast {psi-} {PIN+} cells overproducing Sup35 can exhibit nonsense suppression without generation of a stable {PSI+} . Here we show that in such cells most of Sup35 represents amyloid polymers, though remaining Sup35 monomer is sufficient for normal translation termination . Presence of these polymers strictly depends on {PIN+}, suggesting that their maintenance relies on efficient generation de novo, rather than inheritance . Sup35 polymers contain Rnq1, confirming a hypothesis that Rnq1 polymers seed Sup35 polymerization . About 10 percents of cells overproducing Sup35 form colonies on medium selective for suppression which suggests that the proportion of Sup35 monomers to polymers varies between cells of transformants allowing selection of cells deficient for soluble Sup35 . A hybrid Sup35 with the N-terminal domain replaced for 66 glutamine residues also polymerizes and can cause nonsense suppression when overproduced . The described polymers of these proteins differ from the {PSI+} polymers by poor heritability and very high frequency of the de novo appearance, thus being more similar to amyloids than to prions. Biophys Chem, 2005 Jan 1, 113(1), 41 - 51 Single-molecule detection of yeast cytochrome c by Surface-Enhanced Raman Spectroscopy; Delfino I et al.; The giant enhancement of Raman signal near silver colloidal nanoparticles is exploited to study the Raman spectrum of Cytochrome c from Saccharomyces cerevisiae (Yeast Cytochrome c-YCc) in the limit of single-molecule . The investigation is performed on proteins both in solution and immobilised onto a glass slide using a quasi resonant laser line as exciting source with low excitation intensity . In both cases, spectra acquired at different times exhibit dramatic temporal fluctuations in both the total spectrum and in the specific line intensity, even though averaging of several individual spectra reproduces the main Raman features of bulk YCc . Analysis of the spectral intensity fluctuations from solutions reveals a multimodal distribution of some specific Raman lines, consistent with the approaching of single molecule regime . Among other results, the statistical analysis of the spectra from immobilised samples seems to indicate dynamical processes involving the reorientational of the heme with respect to the metal surface. EMBO J . 2004 Dec 16; {Epub ahead of print} A novel gene amplification system in yeast based on double rolling-circle replication; Watanabe T et al.; Gene amplification is involved in various biological phenomena such as cancer development and drug resistance . However, the mechanism is largely unknown because of the complexity of the amplification process . We describe a gene amplification system in Saccharomyces cerevisiae that is based on double rolling-circle replication utilizing break-induced replication . This system produced three types of amplification products . Type-1 products contain 5-7 inverted copies of the amplification marker, leu2d . Type-2 products contain 13 to approximately 100 copies of leu2d (up to approximately 730 kb increase) with a novel arrangement present as randomly oriented sequences flanked by inverted leu2d copies . Type-3 products are acentric multicopy minichromosomes carrying leu2d . Structures of type-2 and -3 products resemble those of homogeneously staining region and double minutes of higher eukaryotes, respectively . Interestingly, products analogous to these were generated at low frequency without deliberate DNA cleavage . These features strongly suggest that the processes described here may contribute to natural gene amplification in higher eukaryotes. Nature, 2004 Dec 23, 432(7020), 1054 - 8 The transcription factor Ifh1 is a key regulator of yeast ribosomal protein genes; Wade JT et al.; Ribosomal protein (RP) genes in eukaryotes are coordinately regulated in response to growth stimuli and environmental stress, thereby permitting cells to adjust ribosome number and overall protein synthetic capacity to physiological conditions . Approximately 50% of RNA polymerase II transcription is devoted to RP genes . The transcriptional regulator Rap1 binds most yeast RP promoters, and Rap1 sites are important for coordinate regulation of RP genes . However, Rap1 is not the specific regulator that controls RP transcription because it also functions as a repressor, and many Rap1-activated promoters are not coordinately regulated with RP promoters . Here we show that the transcription factors Fhl1 and Ifh1 associate almost exclusively with RP promoters; association depends on Rap1 and (to a lesser extent) a DNA element at many RP promoters . Ifh1 is recruited to promoters via the forkhead-associated (FHA) domain of Fhl1; the level of Ifh1 associated with RP promoters determines the level of transcription; and environmental stress causes a marked reduction in the association of Ifh1, but not Fhl1 or Rap1 . Thus, Ifh1 association with promoters is the key regulatory step for coordinate expression of RP genes. Analyst, 2005 Jan, 130(1), 35 - 7 Epub 2004 Nov 22. Identification of selenomethionine in selenized yeast using two-dimensional liquid chromatography-mass spectrometry based proteomic analysis; McSheehy S et al.; Selenium-enriched yeast has been commonly used as a nutritional supplement . Here we describe a protocol used to investigate the metabolic fate of inorganic selenium in yeast . We provide definitive, mass spectrometry based evidence for the non-specific incorporation of selenomethionine in the yeast proteome involving the replacement of about 30% of all methionine with selenomethionine. RNA . 2004 Dec 21; {Epub ahead of print} RNA-protein interactions in the yeast three-hybrid system: Affinity, sensitivity, and enhanced library screening; Hook B et al.; The yeast three-hybrid system has become a useful tool in analyzing RNA-protein interactions . An RNA sequence is tested in combination with an RNA-binding protein linked to a transcription activation domain (AD) . A productive RNA-protein interaction activates a reporter gene in vivo . The system has been used to test candidate RNA-protein pairs, to isolate mutations in each interacting partner, and to identify proteins that bind a given RNA sequence . However, the relationship between reporter gene activation and in vitro affinity of an RNA-protein interaction has not been examined systematically . This limits interpretation of the data and complicates the development of new strategies . Here, we analyze several key parameters of the three-hybrid system, using as a model the interaction of a PUF protein, FBF-1, with a range of RNA targets . We compare activation of two reporter genes as a function of the in vitro affinity of the interaction . HIS3 and LacZ expression levels are directly related to affinity over a 10-fold range of Kd . Expression of the reporter genes also is directly related to the abundance of the activation domain fusion protein . We describe a new yeast strain, YBZ1, that simplifies screening of cDNA/AD libraries . This strain possesses a tandem, head-to-tail dimer of a high-affinity variant of MS2 coat protein, fused to a monomer of the LexA DNA-binding protein . We show that the use of this strain in cDNA library screens increases the number of genuine, sequence-specific positives detected, and at the same time reduces the background of false, RNA-independent positives. RNA . 2004 Dec 21; {Epub ahead of print} Nonsense-associated alternative splicing of T-cell receptor {beta} genes: No evidence for frame dependence; Mohn F et al.; Mutations that generate premature translation-termination codons (PTCs) often result in production of alternatively spliced mRNAs . While in many cases, the PTC-causing mutation was found to affect splicing directly by disrupting an exonic splicing enhancer, induction of alternative splicing of TCR-betapre-mRNA has been reported to be specific for mutations that prematurely terminate the open reading frame . During testing of a cyto-nuclear feedback model that would have explained how cytoplasmic translation could influence nuclear splicing of TCR-betatranscripts, control experiments questioned the frame dependence of the nonsense-associated altered splicing (NAS) of TCR-betapre-mRNA . A subsequent detailed analysis of alternatively spliced TCR-betamRNA expressed from different minigene constructs with nonsense, silent, or frame-shift mutations at various positions revealed no correlation between truncation of the reading frame and production of alternatively spliced mRNA . Our study thus contradicts the previously reported PTC specificity of TCR-betaNAS and points out the need for systematically testing the PTC specificity in other cases where NAS has been observed. Proc Natl Acad Sci U S A, 2005 Jan 4, 102(1), 51 - 6 Epub 2004 Dec 21. The crystal structure of yeast copper thionein: the solution of a long-lasting enigma; Calderone V et al.; We report here the crystal structure of yeast copper thionein (Cu-MT), determined at 1.44-A resolution . The Cu-MT structure shows the largest known oligonuclear Cu(I) thiolate cluster in biology, consisting of six trigonally and two digonally coordinated Cu(I) ions . This is at variance with the results from previous spectroscopic determinations, which were performed on MT samples containing seven rather than eight metal ions . The protein backbone has a random coil structure with the loops enfolding the copper cluster, which is located in a cleft where it is bound to 10 cysteine residues . The protein structure is somewhat different from that of Ag(7)-MT and similar, but not identical, to that of Cu(7)-MT . Besides the different structure of the metal cluster, the main differences lie in the cysteine topology and in the conformation of some portions of the backbone . The present structure suggests that Cu-MT, in addition to its role as a safe depository for copper ions in the cell, may play an active role in the delivery of copper to metal-free chaperones. Syst Appl Microbiol, 2004 Nov, 27(6), 661 - 71 Trichosporon mycotoxinivorans sp . nov., a new yeast species useful in biological detoxification of various mycotoxins; Molnar O et al.; A yeast strain isolated from the hindgut of the lower termite Mastotermes darwiniensis (Mastotermitidae) was found to represent a new member of the genus Trichosporon . Trichosporon mycotoxinivorans is closely related to T . loubieri on the basis of the phylogenetic trees based on the D1/D2 region of 26S rDNA, an approx . 600 bp fragment of the 18S rDNA and both ITS regions . However, the two species differ at nine positions in the D1/D2 region of 26S rDNA . The IGS1 region of T . mycotoxinivorans is 401 bp long . T . mycotoxinivorans is distinguished from T . loubieri by its ability to assimilate inulin and galactitol, and its inability to grow at 40 degrees C . The name of this newly isolated strain refers to an important characteristics of T . mycotoxinivorans to detoxify mycotoxins such as ochratoxin A and zearalenone . Therefore this strain can be used for the deactivation of the respective mycotoxins in animal feeds. Wei Sheng Yan Jiu, 2004 Sep, 33(5), 565 - 9 {Safety assessment of GM yeast feed additive with cecropin CAD gene}; Deng P et al.; OBJECTIVE: To evaluate the safety of GM yeast feed additive with cecropin CAD and to study and set up a model of Safety assessment for GM feed and detecting method . METHODS: To ensure the safety of the GM products, it has been done that to detect and value the safety of receptor organisms and expression products of extrinic gene, the genetic stability of biologic properties of genomic modified yeast feed and condition of transfer and cumulation of anti-bacterial peptide and its products in circumstance and the feeded animals . RESULT AND CONCLUSION: The receptor animals and expression products of extrinic gene are safe, and the genomic modified products have steady genetic characters . The cectopin CAD neither cumulates in feeded animal nor releases into environment . The genomic modified feed additive is safe. Cell Cycle . 2005 Jan 29;4(1) {Epub ahead of print} A Transcriptional Pathway for Cell Separation in Fission Yeast; Bahler J; Numerous genes are transcriptionally activated and repressed in a cell cycle-dependent manner . We have recently reported the global gene expression program during the cell cycle in fission yeast (S . pombe) . Among the periodically expressed fission yeast genes, a large proportion shows peak transcript levels during mitosis . Many of these genes are regulated by a transcriptional cascade involving two transcription factors: the forkhead protein Sep1p which activates the zinc finger protein Ace2p . A main function of the Sep1p-Ace2p transcriptional pathway is to trigger the separation of daughter cells after cytokinesis . Absence of Sep1p, Ace2p, or some of their target genes leads to a hyphal-like growth pattern with chains of connected cells . Yeast cells probably evolved from filamentous fungi . It is possible that the Sep1p-Ace2p pathway contributed to the emergence of proliferation through single cells, and that this regulatory pathway can still be modulated to adjust growth modes depending on environmental conditions . Here, various properties of the Sep1p-Ace2p transcriptional pathway and mechanisms for cell separation are discussed in the context of recent findings. Genetics, 2004 Dec, 168(4), 2421 - 4 Does chromosome size affect map distance and genetic interference in budding yeast? Turney D, de Los Santos T, Hollingsworth NM. The hypothesis that chromosome size affects the rate and distribution of meiotic crossovers in budding yeast was tested . Map distance and interference were measured in the same genetic intervals present on either small (340 and 508 kb) or large (917 and 1085 kb) chromosomes, respectively . No differences were observed. Genetics, 2004 Dec, 168(4), 1827 - 41 Genetic analysis of yeast yip1p function reveals a requirement for Golgi-localized rab proteins and rab-Guanine nucleotide dissociation inhibitor; Chen CZ et al.; Yip1p is the first identified Rab-interacting membrane protein and the founder member of the YIP1 family, with both orthologs and paralogs found in all eukaryotic genomes . The exact role of Yip1p is unclear; YIP1 is an essential gene and defective alleles severely disrupt membrane transport and inhibit ER vesicle budding . Yip1p has the ability to physically interact with Rab proteins and the nature of this interaction has led to suggestions that Yip1p may function in the process by which Rab proteins translocate between cytosol and membranes . In this study we have investigated the physiological requirements for Yip1p action . Yip1p function requires Rab-GDI and Rab proteins, and several mutations that abrogate Yip1p function lack Rab-interacting capability . We have previously shown that Yip1p in detergent extracts has the capability to physically interact with Rab proteins in a promiscuous manner; however, a genetic analysis that covers every yeast Rab reveals that the Rab requirement in vivo is exclusively confined to a subset of Rab proteins that are localized to the Golgi apparatus. J Biol Chem . 2004 Dec 17; {Epub ahead of print} Conditional osmotic stress in yeast: A system to study transport through aquaglyceroporins and osmostress signaling; Karlgren S et al.; The accumulation and transport of solutes are hallmarks of osmoadaptation . In this study we have employed the inability of the Saccharomyces cerevisiae gpd1 gpd2 mutant both to produce glycerol and to adapt to high osmolarity in order to study solute transport through aquaglyceroporins and the control of osmostress-induced signaling . High levels of different polyols, including glycerol, inhibited growth of the gpd1 gpd2 mutant . This growth inhibition was suppressed by expression of the hyperactive allele Fps1-1 of the osmogated yeast aquaglyceroporin, Fps1 . The degree of suppression correlated with the relative rate of transport of the different polyols tested . Transport studies in secretory vesicles confirmed that Fps1-1 transports polyols at increased rates compared with wild type Fps1 . Importantly, wild type Fps1 and Fps1-1 showed similarly low permeability for water . The growth defect on polyols in the gpd1 gpd2 mutant was also suppressed by expression of a heterologous aquaglyceroporin, rat AQP9 . We surmised that this suppression was due to polyol influx causing the cells to passively adapt to the stress . Indeed, when aquaglyceroporin-expressing gpd1 gpd2 mutants were treated with glycerol, xylitol or sorbitol the osmosensing HOG pathway was activated and the period of activation correlated with the apparent rate of polyol uptake . This observation supports the notion that deactivation of the HOG pathway is closely coupled to osmotic adaptation . Taken together, our "conditional" osmotic stress system facilitates studies on aquaglyceroporin function and reveals features of the osmosensing and signaling system. J Biol Chem . 2004 Dec 20; {Epub ahead of print} Yeast flavohemoglobin, a nitric oxide oxidoreductase, is located both in the cytosol and mitochondrial matrix; Cassanova N et al.; Yeast flavohemoglobin, YHb, encoded by the nuclear gene, YHB1, has been implicated in both the oxidative and nitrosidative stress responses in Saccharomyces cerevisiae . Previous studies have shown that expression of YHB1 is optimal in normoxic or hyperoxic conditions yet respiring yeast cells have low levels of reduced YHb pigment, detected by carbon monoxide (CO) photolysis difference spectroscopy of glucose-reduced cells . Here, we have addressed this apparent discrepancy by determining the intracellular location of the YHb protein and analyzing the relationships among respiration, YHb level, and intracellular location . We have found that although intact respiration-proficient cells lack a YHb CO-spectral signature, cell extracts from these cells have both a YHb CO-spectral signature and nitric oxide (NO) consuming activity . This suggests either that YHb can not be reduced in vivo or that YHb heme is maintained in an oxidized state in respiring cells . By using an anti-YHb antibody, CO-difference spectroscopy, and by measuring NO consumption we have found that YHb localizes to two distinct intracellular compartments, the mitochondrial matrix and the cytosol, in respiring cells . Moreover, we have found that the distribution of YHb between these two compartments is affected by the presence or absence of oxygen and by the mitochondrial genome . These findings suggest that YHb functions in oxidative stress indirectly by consuming NO, which inhibits mitochondrial respiration and leads to enhanced production of reactive oxygen species, and that cells can regulate intracellular distribution of YHb in accordance with this function. J Biol Chem . 2004 Dec 20; {Epub ahead of print} Genome wide analysis reveals inositol, not choline, as the major effector of Ino2p-Ino4p and unfolded protein response target gene expression in yeast; Jesch SA et al.; In the yeast Saccharomyces cerevisiae the transcription of many genes encoding enzymes of phospholipid biosynthesis are repressed in cells grown in the presence of the phospholipid precursors inositol and choline . A genome-wide approach using cDNA microarray technology was utilized to profile the changes in the expression of all genes in yeast that respond to the exogenous presence of inositol and choline . We report that the global response to inositol is completely distinct from the effect of choline . Whereas the effect of inositol on gene expression was primarily repressing, the effect of choline on gene expression was activating . Moreover, the combination inositol and choline increased the number of repressed genes compared to inositol alone and enhanced the repression levels of a subset of genes that responded to inositol . In all, 110 genes were repressed in the presence of inositol and choline . Two distinct sets of genes exhibited differential expression in response to inositol or the combination of inositol and choline in wild type cells . One set of genes contained the UASINO sequence and were bound by Ino2p and Ino4p . Many of these genes were also negatively regulated by OPI1, suggesting a common regulatory mechanism for Ino2p, Ino4p, and Opi1p . Another non-overlapping set of genes were coregulated by the unfolded protein response pathway, an ER-localized stress response pathway, but were not dependent on OPI1 and did not show further repression when choline was present together with inositol . These results suggest that inositol is the major effector of target gene expression, while choline plays a minor role. J Biol Chem . 2004 Dec 14; {Epub ahead of print} Native replication intermediates of the yeast 20 S RNA virus have a single-stranded RNA backbone; Fujimura T et al.; 20 S RNA virus is a positive strand RNA virus found in Saccharomyces cerevisiae . The viral genome (2.5 kb) only encodes its RNA polymerase (p91) and forms a ribonucleoprotein complex with p91 in vivo . A lysate prepared from 20 S RNA-induced cells showed an RNA polymerase activity that synthesized the positive strands of viral genome . When in vitro products, after phenol extraction, were analyzed in time course, radioactive nucleotides were first incorporated into double-stranded RNA intermediates and then chased out to the final single-stranded RNA products . The positive and negative strands in these dsRNA intermediates were non-covalently associated and the release of the positive strand products from the intermediates required a net RNA synthesis . We found, however, that these dsRNA intermediates were an artifact caused by phenol-extraction . Native replication intermediates had an ssRNA backbone as judged by RNase sensitivity experiments and they migrated distinctly from a dsRNA form in non-denaturing gels . Upon completion of RNA synthesis, positive strand RNA products as well as negative strands templates were released from replication intermediates . These results indicate that the native replication intermediates consists of a positive strand less than unit length and a negative strand template loosely associated, probably through the RNA polymerase p91 . Therefore, W, a dsRNA form of 20 S RNA that accumulates in yeast cells grown at 37 masculineC is not an intermediate in the 20 S RNA replication cycle, but a byproduct. Mech Ageing Dev, 2005 Jan, 126(1), 17 - 21 Large-scale identification in yeast of conserved ageing genes; Kaeberlein M et al.; Recent advances have suggested the existence of phylogenetically conserved pathways regulating ageing in eukaryotes . At least two of these "public" longevity-determining pathways appear to have been evolutionarily conserved from yeast through mammals . We have developed a high-throughput, genome-wide approach to identify a large fraction of the non-essential, single-gene deletion mutations that confer increased longevity in yeast . The identification and characterization of conserved genes that regulate the ageing process across eukaryotic species is likely to result in an improved understanding of the causes of human ageing and provide potential therapeutic targets for drug discovery. Mech Ageing Dev, 2005 Jan, 126(1), 11 - 6 Analysis of gene expression profile in yeast aging chronologically; Fabrizio P et al.; The use of simple model systems such as Saccharomyces cerevisiae and Caenorhabditis elegans has played a primary role in the identification of proteins and pathways that regulate the aging process in eukaryotes . Recent findings have shown that analogous pathways regulate aging in higher eukaryotes and suggest a conserved origin for the molecular mechanisms that regulate stress-resistance and longevity . Genomics approaches that allow the simultaneous monitoring of the expression of thousands of genes are beginning to reveal the complexity of the molecular changes required to extend life span . Here we describe how analysis of the gene expression profiles of wild-type and long-lived yeast aging chronologically can be used to identify proteins that increase stress-resistance and longevity . We also discuss a novel genomics method for the identification of chronologically long-lived yeast mutants. Biochemistry, 2004 Dec 28, 43(51), 16254 - 62 Yeast frataxin solution structure, iron binding, and ferrochelatase interaction; He Y et al.; The mitochondrial protein frataxin is essential for cellular regulation of iron homeostasis . Although the exact function of frataxin is not yet clear, recent reports indicate the protein binds iron and can act as a mitochondrial iron chaperone to transport Fe(II) to ferrochelatase and ISU proteins within the heme and iron-sulfur cluster biosynthetic pathways, respectively . We have determined the solution structure of apo yeast frataxin to provide a structural basis of how frataxin binds and donates iron to the ferrochelatase . While the protein's alpha-beta-sandwich structural motif is similar to that observed for human and bacterial frataxins, the yeast structure presented in this report includes the full N-terminus observed for the mature processed protein found within the mitochondrion . In addition, NMR spectroscopy was used